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DNA: Structure, Dynamics and Recognition

DNA: Structure, Dynamics and Recognition. L4: DNA deformation. Les Houches 2004. BASE PAIR OPENING. Bond vibrations 1 fs (10 -15 s) Sugar repuckering 1 ps (10 -12 s) DNA bending 1 ns (10 -9 s) Domain movement 1  s (10 -6 s) Base pair opening 1 ms (10 -3 s)

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DNA: Structure, Dynamics and Recognition

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  1. DNA: Structure, Dynamics and Recognition L4: DNA deformation Les Houches 2004

  2. BASE PAIR OPENING

  3. Bond vibrations 1 fs (10-15 s) Sugar repuckering 1 ps (10-12 s) DNA bending 1 ns (10-9 s) Domain movement 1 s (10-6 s) Base pair opening 1 ms (10-3 s) Transcription 2.5 ms / nucleotide Protein synthesis 6.5 ms / amino acid Protein folding ~ 10 s RNA lifetime ~ 300 s Biological time scale

  4. Enzymatic base chemistry

  5. S S HN3 imino proton Adenine-Thymine base pair

  6. S S HN1 imino proton Guanine-Cytosine base pair

  7. GC 15-25 ms AT 5-10 ms C G C A A G A A G C G * * 4 1 1 23 4 5 4 * * Base opening lifetimes

  8. Base pair lifetimes (ms) 15°C T T T T A A A A 1 17 19 4 4 19 17 1 A A A A T T T T 60 100 100 65 65 100 100 60 Leroy et al. Biochemistry 27, 1988, 8894 A4T4 versus T4A4

  9. B-DNA - 2ns dynamic trajectory

  10. Free energy calculations using restrained opening Guidice et al. ChemPhysChem 2, 2001, 673 Varnai & Lavery J. Am. Chem. Soc. 124, 2002, 7272

  11. BIASED PROBABILITY HISTOGRAM Nw  P*i(q) exp [Vi(q)] i =1 Pi(q)   Nw  ni exp [Fi(q)Vi(q)] i =1 Nw Fi(q) kT ln  Pi(q) i =1 N(q) FREE ENERGY PROFILE W(q) Reaction coordinate (q) WHAM

  12. G A G A G A G A G A G A G C T C T C T C T C T C T C B-DNA oligonucleotide studied

  13. Extraction d’une base de l’ADN

  14. Closed AT pair

  15. Adenine –50° (minor)

  16. Adenine –100° (minor)

  17. Adenine +50° (major)

  18. Adenine +100° (major)

  19. Free energy curves for base opening

  20. T G Imino proton accessibility (Å2)

  21. Dq Thymine (°) DqAdenine (°) Dq Adenine (°) Dq Thymine (°) Base movements are coupled

  22. T A-tract T Ref Dq Sequence effects on opening: A-tracts

  23. G T q < -50° -50° < q < +50° q > +50° Bending amplitude (°)

  24. A word of warning!

  25. BASE FLIPPING

  26. Hha1 methyltransferaseKlimašauskas et al. Cell 76 (1994) 357

  27. Minor groove  Major groove

  28. +160° opening -200° opening

  29. Backbone rearrangements

  30. Backbone rearrangements

  31. SUPERCOILING

  32. DNA supercoiling (circular plasmid)

  33. L = linking number = number of strand crossings T = twist = number of turns of double helix W = writhe = number of helix crossovers L = T + W s = supercoiling density = (L – L0) / L0 = DL / L0 typically s ~ -0.06 (1 crossing less per 17 turns) DNA supercoiling

  34. Linking number (L or Lk) – a topological constant

  35. Low force High force Twist (T) versus Writhe (W)

  36. L = T + W

  37. L.H. R.H. Interwound and toroidal forms of a negatively supercoiled plasmid

  38. Ethidium bromide intercalates into DNA and reduces its twist by ~26°

  39. Effect of an intercalator on a negatively supercoiled plasmid

  40. Topoisomerase I - single strand cuts - releases negative supercoiling Topoisomerase II - double strand cuts (eukaryotes) - releases negative supercoiling Topo II (gyrase) - generates negative supercoiling (prokaryotes) - consumes ATP Reverse gyrase - generates positive supercoiling (thermophiles) Topoisomerases

  41. Topoisomerase I – single strand cuts

  42. Topoisomerase II – double strand cuts

  43. Topo II (gyrase)DNA wrapping

  44. DNA packed on nucleosomes

  45. Nucleosome – schematic view

  46. EXTREME DEFORMATIONS

  47. DNA stretching Cluzel et al. Science 271, 1996, 792

  48. 70 pN phase transition

  49. S-DNA: fibre and ribbon forms

  50. Greenall et al. J. Mol. Biol. 2001, 305, 669 Rise ~ 5.6 Å Helix spacing ~ 13 Å Fibre diffraction of stretched DNA

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